G-protein coupled receptors desensitize in the prolonged presence of agonist, a process that involves phosphorylation and arrestin binding. Normal desensitization of the GPCR rhodopsin, the rod photoreceptor visual pigment, controls the kinetics of responses to single photons, and relies on a full complement of six phosphorylation sites on the rhodopsin C-terminus (three serines and three threonines). How these sites facilitate arrestin binding at the molecular level remains unclear, so we compared single-photon responses (SPRs) from mouse rods expressing rhodopsin with only the native serine or threonine residues. We found that, while SPRs produced by rhodopsin with only threonine residues remain relatively stereotyped in shape, serine-only rhodopsin generates prolonged step-like events that abruptly terminate randomly in time, with a median lifetime of ~2.9 seconds. Genetically lowering arrestin expression further prolonged the responses, suggesting that the step-like recovery reflects arrestin binding. Serine phosphorylation alone is thus insufficient for normal rhodopsin desensitization, so that serine and threonine phosphorylation sites play distinct yet complementary roles in desensitization of a GPCR.